Biochem. J. (2014) 464, 85–98 (Printed in Great Britain) doi:10.1042/BJ20131609 85

Specific aromatic foldamers potently inhibit spontaneous and seededAβ42 and Aβ43 fibril assemblyKatelyn M. Seither*, Heather A. McMahon*, Nikita Singh*, Hejia Wang*, Mimi Cushman-Nick*†‡, Geronda L. Montalvo*§,William F. DeGrado‡ and James Shorter*†§1

*Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, 805b Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA19104, U.S.A.†Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 805b Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104, U.S.A.‡Department of Pharmaceutical Chemistry, University of California, San Francisco, CVRI-MC Box 3122, San Francisco, CA 94158-9001 U.S.A.§Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 805b Stellar-Chance Laboratories, 422 Curie Boulevard,Philadelphia, PA 19104, U.S.A.

Amyloid fibrils are self-propagating entities that spread pathologyin several devastating disorders including Alzheimer’s disease(AD). In AD, amyloid-β (Aβ) peptides form extracellular plaquesthat contribute to cognitive decline. One potential therapeuticstrategy is to develop inhibitors that prevent Aβ misfoldinginto proteotoxic conformers. Here, we design specific aromaticfoldamers, synthetic polymers with an aromatic salicylamide(Sal) or 3-amino benzoic acid (Benz) backbone, short length(four repetitive units), basic arginine (Arg), lysine (Lys) orcitrulline (Cit) side chains, and various N- and C-terminalgroups that prevent spontaneous and seeded Aβ fibrillization. Ac-Sal-(Lys-Sal)3-CONH2 and Sal-(Lys-Sal)3-CONH2 selectivelyinhibited Aβ42 fibrillization, but were ineffective against Aβ43,an overlooked species that is highly neurotoxic and frequentlydeposited in AD brains. By contrast, (Arg-Benz)4-CONH2 and

(Arg-Sal)3-(Cit-Sal)-CONH2 prevented spontaneous and seededAβ42 and Aβ43 fibrillization. Importantly, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibited formation of toxic Aβ42 and Aβ43 oligomersand proteotoxicity. None of these foldamers inhibited Sup35prionogenesis, but Sal-(Lys-Sal)3-CONH2 delayed aggregationof fused in sarcoma (FUS), an RNA-binding protein with aprion-like domain connected with amyotrophic lateral sclerosisand frontotemporal dementia. We establish that inhibitors ofAβ42 fibrillization do not necessarily inhibit Aβ43 fibrillization.Moreover, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibits formation oftoxic Aβ conformers and seeding activity, properties that couldhave therapeutic utility.

Key words: Alzheimer’s disease, amyloid, Aβ42 (amyloid-β 42),Aβ43 (amyloid-β 43), foldamer, protein misfolding.

INTRODUCTION

Protein misfolding can be fatal [1,2]. Proteins misfold fromsoluble species into highly stable, cross-β amyloid fibrils inAlzheimer’s disease (AD) and several other neurodegenerativediseases [1,2]. One strategy to combat these disorders is todevelop small molecules that inhibit amyloidogenesis and preventtoxic protein misfolding [3–6]. Although daunting challengesface potential small molecule inhibitors of amyloidogenesis [7],they are beginning to reach the clinic. Indeed, tafamidis,a small molecule inhibitor of transthyretin amyloidogenesistreats familial amyloid polyneuropathy, a rare but deadlydisease [8,9].

Here, we focus on amyloid-β (Aβ) peptides, Aβ42 and Aβ43,which form amyloid fibrils and accumulate in extracellularplaques that are a hallmark of AD [10–16]. AD is a progressiveneurodegenerative disease and the most common cause ofdementia worldwide [12]. Aging is a significant risk factor forAD and there are no effective therapies [11]. In Aβ biogenesis,the full-length transmembrane amyloid precursor protein (APP)undergoes sequential cleavage by β- and γ -secretase, resultingin peptides that are 38–43 amino acids in length [10,12]. Aβ42and Aβ40 are most commonly associated with AD pathology

[10–12]. Aβ40 is a more benign, perhaps even neuroprotectivespecies [17,18], which slowly assembles into amyloid fibrils. Bycontrast, Aβ42 oligomerizes and fibrillizes more rapidly due totwo additional C-terminal residues that introduce additional stericzipper hexapeptides that drive assembly [19–21].

Although Aβ peptides longer than Aβ42 are found in AD, theyare not a major species and their pathogenic role has been ignored.Recently, this view has changed. Aβ43 is a potent contributorto neurotoxicity in AD [13–15]. Aβ43 contains an additionalthreonine residue at the C-terminal end and fibrillizes morerapidly than Aβ42 [13]. Aβ43 is more abundant in insolublefractions than Aβ40 in AD and its presence in senile plaquesis directly correlated with cognitive decline [13–16]. Specificinhibitors of Aβ43 misfolding have not been identified and itis unclear whether inhibitors of Aβ42 misfolding will also inhibitAβ43 misfolding.

Aβ monomers form amyloid via nucleated conformationalconversion [22]. First, a subpopulation of Aβ monomers formsmolten oligomers, which gradually rearrange into amyloidogenicoligomers that nucleate cross-β fibrils [22,23]. Rearrangement israte limiting and causes the lag phase of spontaneous fibrillization[22]. During lag phase, Aβ forms diverse oligomeric species,which can be highly toxic [21,24–27]. Upon nucleation, fibrils

Abbreviations: AD, Alzheimer’s disease; Aβ, amyloid-β; Benz, 3-amino benzoic acid; DCM, dichloromethane; DIEA, di-isopropylethylamine; DMEM,Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; EGCG, ( − )-epigallocatechin-3-gallate; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; LDH,lactate dehydrogenase; MeOH, methanol; NM, N-terminal and middle domains of Sup35; Sal, salicylamide; TEV, tobbaco etch virus; ThT, thioflavin-T.

1 To whom correspondence should be addressed (email jshorter@mail.med.upenn.edu).

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86 K. M. Seither and others

Figure 1 Overview of aromatic foldamer structure

The core foldamer structure is shown in the dashed box, which can be decorated with different moieties at X-, R-, Y- and Z-positions indicated on the periphery. Foldamers possess an aromatic Salor Benz backbone (Y = OMe or H), Arg, Lys or Cit side chains (R = Arg, Lys or Cit), short length (two to four repetitive units) and various N- (X = NH2 or Ac) and C- (Z = NH2, OH, OMe or β-Ala)terminal groups.

rapidly grow via their self-templating ends, which convert Aβconformers into the cross-β conformation [20,28]. When coupledto fibril fragmentation, this ‘seeding’ activity enables Aβ fibrils tobecome self-propagating agents that transmit pathology anddisease [1,29–31]. Aβ fibrils also provide catalytic surfaces for‘secondary’ nucleation events distinct from fibril elongation [32–34]. Here, lateral Aβ fibril surfaces convert Aβ monomers intotoxic oligomers [32–34]. Thus, formation of toxic oligomers andfibrils is intimately linked [32–34]. These secondary nucleationevents also help explain Aβ assembly kinetics [32–34]. Aβforms different cross-β fibril structures termed ‘strains’, whichcan differ in toxicity and cause distinct brain pathology [35–38]. Aβ fibrils are usually less toxic than pre-amyloid oligomers[21,39]. However, Aβ fibrils also display toxicity [6,35,36,39].A key challenge is to manipulate Aβ assembly in a mannerthat depopulates toxic conformers [7]. Agents that inhibit seededassembly hold promise for preventing the spread of Aβ pathologyin AD.

Numerous potential inhibitors of Aβ misfolding havebeen explored, including small molecules, peptides, molecularchaperones, protein disaggregases and antibodies [3,6,39–45].In the present study, we explore a different strategy bypursuing foldamers; non-biological discrete chain molecules thatlack a canonical peptide backbone but can fold into specificstructures [46]. Foldamers have been utilized as antimicrobialagents and molecular scaffolds [47–50]. Peptides containing non-natural amino acids, similar to foldamers, have been useful forunderstanding the misfolding of various amyloidogenic peptides[42,51–53]. Foldamers have several advantageous properties thatcould make them a valuable class of amyloid inhibitors. Due totheir semi-rigid backbone, foldamers can assume an organizedconformation at low entropic cost with relatively few monomericunits [50,54]. Compared with α peptides, foldamers have greaterthermodynamic stability and resist proteases. Furthermore,foldamers of varying lengths with diverse side chains and 3Dshapes can be synthesized. These features enable foldamer designfor interaction with diverse biological targets [47–50,55]. In thepresent study, we explore aromatic foldamers as antagonists ofAβ42 and Aβ43 amyloidogenesis.

MATERIALS AND METHODS

Generation of soluble and fibrillar Aβ42 and Aβ43

To produce monomeric Aβ, synthetic lyophilized Aβ42 orAβ43 (W.M. Keck Facility, Yale University) was dissolvedin 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) at 2 mg/ml.HFIP was removed by drying in a speed vacuum for 30 min.The resulting peptide film was dissolved in DMSO to 1 mM.Aβ42 or Aβ43 fibrils for seeding experiments were prepared bydiluting monomerized Aβ42 or Aβ43 in KHMD (150 mM KCl,40 mM Hepes–KOH pH 7.4, 20 mM MgCl2 and 1 mM DTT) to10 μM. This solution was incubated at 37 ◦C for 3–5 days withagitation (700 r.p.m.) in an Eppendorf Thermomixer. For seedingexperiments, preformed fibrils were briefly sonicated or vortex-mixed prior to use. We also prepared Aβ42 or Aβ43 using aprotocol that avoids DMSO. Thus, Aβ42 or Aβ43 was dissolvedin HFIP followed by evaporation of the solvent to dryness [56].Dry peptide films were dissolved in a minimal volume of 60 mMNaOH followed by dilution with deionized water and sonicationfor 1 min using a bath sonicator. Peptides were diluted to 0.2 mMby adding an equal volume of 20 mM sodium phosphate buffer(PB, Sigma), pH 8 plus 0.2 mM EDTA (PBE). Samples werecentrifuged at 16000 g for 3 min and subjected to Superdex 75gel filtration in PBE to remove residual solvent.

Foldamers

Foldamers (Lys-Sal)4-CONH2, (Arg-Benz)4-CONH2, (Lys-Sal)4-COMe, (Lys-Sal)4-COOH, (Lys-Sal)4-COβAla, Ac-(Lys-Sal)3-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2

(where Sal is salicylamide and Benz is 3-amino benzoic acid) werefrom PolyMedix and were dissolved in TBS (50 mM Tris/HClpH 7.4, 150 mM NaCl) to obtain concentrated stock solutions.Foldamers (Cit-Sal)4-CONH2, (Arg-Sal)2-(Cit-Sal)-(Arg-Sal)-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, (Cit-Sal)2-(Arg-Sal)-(Cit-Sal)-CONH2, (Cit-Sal)-(Arg-Sal)-(Cit-Sal)2-CONH2 and (Arg-Sal-Cit-Sal)2-CONH2 were also from PolyMedix. Thesefoldamers were dissolved in 1:1 TBS/DMSO to obtain

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Specific aromatic foldamers antagonize Aβ amyloidogenesis 87

Figure 2 Nomenclature and structure of aromatic foldamers

Three-letter amino acid nomenclature is used to indicate the side chain (Lys, Arg or Cit) and the Sal or Benz backbone is indicated. N- (Ac) and C- (NH2, OH, OMe or β-Ala) terminal groups are alsoindicated. Foldamers that inhibit spontaneous Aβ42 and Aβ43 fibrillization, (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2, are boxed in black. Foldamers that inhibit spontaneous Aβ42fibrillization but not spontaneous Aβ43 fibrillization, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2, are boxed in grey.

concentrated stocks. Subsequent dilutions were made from thesestocks to appropriate concentrations in KHMD or PBE.

Foldamers (Lys-Sal)2-CONH2, Ac-(Lys-Sal)2-CONH2, Sal-(Lys-Sal)2-CONH2, (Lys-Sal)3-CONH2 and Ac-(Lys-Sal)3-CONH2 were synthesized at room temperature on a 100 μmolscale using rink amide resin (GemScript Corporation, 0.6 mmol/gsubstitution) for support of alternating α- (Bachem) and aromaticamino acids. Resin was swelled in 100% dimethylformamide(DMF, Fisher Scientific) for 1 h, followed by a 30 mindeprotection using 5% piperazine (Sigma–Aldrich) in DMF.

The first residue was coupled to the resin using 3 equiv. ofamino acid, 2.8 equiv. of 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU, GLBiosciences) activator and 7.5 equiv. of di-isopropylethylamine(DIEA, CHEM-IMPEX International), shaking for 1 h at roomtemperature. The resin was washed three times each withDMF, dichloromethane (DCM, Fisher Scientific) and DMF. Thisstep was followed by deprotection (as above). Coupling anddeprotection steps were cycled for the remaining residues ineach respective peptide sequence. After deprotection of the

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Figure 3 (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 inhibit spontaneous Aβ42 fibrillization

(A) Aβ42 (5 μM) was incubated with agitation for 8 h at 25◦C plus or minus the indicated foldamer (10 μM). Aβ42 fibrillization was assessed by ThT fluorescence. Values represent means +− S.E.M.(n = 3–6). A one-way ANOVA with the post-hoc Dunnett’s multiple comparisons test was used to compare Aβ42 alone to each Aβ42 plus foldamer condition (* denotes P < 0.05). Foldamers thatselectively inhibit Aβ42 fibrillization are indicated by grey bars and foldamers that inhibit Aβ42 and Aβ43 fibrillization are indicated by black bars. (B) Aβ42 (5 μM) was incubated with agitationfor 4 h at 25◦C in the absence or presence of the indicated foldamer (10 μM). Aβ42 fibrillization was assessed by EM. Scale bar, 500 nm.

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Specific aromatic foldamers antagonize Aβ amyloidogenesis 89

Figure 4 Effect of inhibitory foldamers on spontaneous Aβ42 fibrillization kinetics

(A–D) Aβ42 (5 μM) was incubated with agitation for 0–8 h at 25◦C in the absence (open circles) or presence of 5 μM (filled triangles), 10 μM (filled squares) or 20 μM (filled circles)(Arg-Benz)4-CONH2 (A), (Arg-Sal)3-(Cit-Sal)-CONH2 (B), Sal-(Lys-Sal)3-CONH2 (C) or Ac-Sal-(Lys-Sal)3-CONH2 (D). Aβ42 fibrillization was assessed by ThT fluorescence. Values representmeans +− S.E.M. (n = 3).

final residue the product was rinsed [three times with DMF,three times with DCM, three times with DMF and three timeswith methanol (MeOH)] and dried with MeOH. This productwas split in half. The first half was re-swelled in DMF andacetylated by incubating the resin in 5% acetic anhydride in 2.5%DIEA and 92.5% DMF for 10 min. This acetylated portion wasrinsed and dried (as above). Next, both halves (one with a N-terminal acetyl and a second with a N-terminal free amide) werecleaved from the resin using a cocktail of 2:2:2:94 H2O/TIS(tri-isopropyl silane)/anisole/TFA (trifluoroacetic acid; Sigma–Aldrich) for 2 h at room temperature. The peptide solution wasfiltered from the resin and precipitated using 1:1 cold ethylether:hexane. The precipitate was dried by lyophilization. Themass and purity of each product was verified by MALDI–TOFMS (Brucker microflex LRF) and analytical HPLC (C18 column).Dried crude foldamer was purified by preparative reverse-phaseHPLC, dried by lyophilization and mass and purity was verifiedas above. All samples were prepared by directly dissolvinglyophilized foldamer into TBS buffer to 2 mM.

Spontaneous and seeded Aβ42, Aβ43 and N-terminal and middledomain of Sup35 (NM) fibrillization

For spontaneous fibrillization, soluble Aβ42 or Aβ43 (1 mM)in DMSO was diluted to 5 μM in KHMD containing25 μM thioflavin-T (ThT) plus or minus foldamer (0–20 μM).For seeded fibrillization, preformed Aβ42 or Aβ43 fibrils(10 μM monomer) were added at a final concentration of0.1 μM (monomer). Alternatively, Aβ42 or Aβ43 were preparedusing just HFIP and were assembled at 5 μM in PBE containing25 μM ThT plus or minus foldamer (20 μM). NM was purified asdescribed [57]. NM (5 μM) was assembled in KHMD containing

25 μM ThT plus or minus foldamer (20 μM). For seededfibrillization, preformed NM fibrils (5 μM monomer) were addedat a final concentration of 0.1 μM (monomer). Reactions wereconducted in 96-well plates and incubated at 25 ◦C in a TECANSafire II plate reader (Tecan USA) for up to 8 h with agitation. ThTfluorescence was measured at the indicated times. The excitationwavelength was 450 nm (5 nm bandwidth) and the emissionwavelength was 482 nm (10 nm bandwidth). ThT fluorescencevalues reported are arbitrary and are normalized to the finalassembly time point of the Aβ alone condition.

FUS aggregation

GST–TEV–FUS was purified as described [58]. Aggregation wasinitiated by addition of tobbaco etch virus (TEV) protease toGST–TEV–FUS (5 μM) plus or minus foldamer (20 μM) inassembly buffer (50 mM Tris/HCl pH 8, 0.2 M trehalose and20 mM glutathione). Aggregation was for 0–90 min at 25 ◦Cwithout agitation in a 96-well plate and was assessed by turbidity(absorbance at 395 nm) using a Tecan Infinite M1000 plate reader[58]. No aggregation occurred unless TEV protease was added toseparate GST from FUS [58]. SDS/PAGE and Coomassie stainingrevealed that foldamers did not inhibit cleavage of GST–TEV–FUS by TEV.

Electron microscopy

Reactions were adhered on to 300-mesh-formvar carbon-coatedEM grids overnight before being negatively stained with 2%uranyl acetate for 2 min and rinsed with milli-Q distilled water.Micrographs were acquired using a JEOL 1010 TEM (Jeol USA).

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Tracking A11-reactive Aβ42 or Aβ43 conformers

The oligomer-specific A11 antibody was used to detect toxicAβ42 or Aβ43 oligomers by ELISA as described [21]. Foldamersdid not cross-react with A11.

Toxicity assays

SH-SY5Y human neuroblastoma cells were maintained inDulbecco’s modified Eagle’s medium (DMEM) plus 10 mMHepes, 10% FBS, 4 mM glutamine, penicillin (200 units/ml)and streptomycin (200 μg/ml) in 5% CO2 at 37 ◦C. Cells weredifferentiated in serum-free DMEM with N2 supplement and10 μM all-trans-retinoic acid before use. Cells were platedat (10000 cells/well) in 96-well plates and grown overnight.Medium was removed and Aβ conformers or controls were addedand cells were incubated for 24 h at 37 ◦C. Toxicity was assessedusing an MTT kit (Tox-1; Sigma) or via lactate dehydrogenase(LDH) release using the CytoTox-ONETM kit (Promega). Toxicityvalues were normalized to the buffer control without Aβ.

RESULTS AND DISCUSSION

Rationale and foldamer design

As potential inhibitors of Aβ42 and Aβ43 amyloidogenesis, weexplored aromatic foldamers (Figures 1 and 2). Some of thesefoldamers were originally synthesized as inhibitors of heparinand are rich in aromatic and positively charged groups [55].They possess an aromatic salicylamide (Sal) or 3-amino benzoicacid (Benz) backbone (Figure 1; Y = OMe or H), lysine (Lys),arginine (Arg) or citrulline (Cit) side chains (Figure 1; R = Lys,Arg or Cit), short length (two to four repetitive units) (Figure 1)and various N- (Figure 1; X = NH2 or COMe [Ac]) and C-(Figure 1; Z = NH2, OH, OMe or β-Ala) terminal groups. Weselected this design for four reasons. First, the aromatic backboneis similar to ones employed by Nowick et al. [42,51–53] inprotein aggregation inhibitors. Secondly, interactions betweenaromatic residues within short amyloidogenic peptides mediatemolecular recognition during fibrillization [59]. Moreover,polyphenols such as ( − )-epigallocatechin-3-gallate (EGCG)inhibit amyloidogenesis and prevent cytotoxicity [57,59–61].Thus, the aromatic foldamer spine might antagonize aromaticinteractions critical for fibrillization. Thirdly, the aromaticfoldamers investigated are approximately the same length (twoto four repetitive units) as steric zipper hexapeptides that formamyloid [19]. Finally, basic side chains, particularly arginine exerthydrotropic effects and prevent protein aggregation [62].

Foldamer inhibition screen

We tested 18 aromatic foldamers (Figure 2) for inhibition ofspontaneous (i.e. in the absence of preformed fibrils) Aβ42fibrillization. The majority of foldamers did not significantlyinhibit Aβ42 fibrillization (Figure 3A). However, (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2

and Ac-Sal-(Lys-Sal)3-CONH2 were strong inhibitors (Figure 2boxed in black or grey; Figures 3A and 3B; Figures 4A–4D).(Arg-Sal)3-(Cit-Sal)-CONH2 was the most potent with an IC50 of∼1.6 μM.

Several important foldamer properties emerge for inhibitionof Aβ42 fibrillization. First, a foldamer must have a backbonewith at least four aromatic units to antagonize Aβ42fibrillization. Thus, (Lys-Sal)2-CONH2, Ac-(Lys-Sal)2-CONH2,Sal-(Lys-Sal)2-CONH2, (Lys-Sal)3-CONH2 and Ac-(Lys-Sal)3-

Figure 5 Sal-(Lys-Sal)3-CONH2 has no effect on NM fibrillization but delaysFUS aggregation

(A) NM (5 μM) was incubated with agitation for 4 h at 25◦C plus or minus the indicated foldamer(20 μM). NM fibrillization was assessed by ThT fluorescence. Values represent means +− S.E.M.(n = 3). A one-way ANOVA with the post-hoc Dunnett’s multiple comparisons test was used tocompare NM alone to each NM plus foldamer condition (* denotes P < 0.05). (B) GST-FUS(5 μM) was incubated in the presence of the indicated foldamer (20 μM) plus TEV protease at25◦C for 0–90 min. Turbidity measurements (absorbance at 395 nm) were taken every minuteto assess aggregation. A representative dataset is shown.

CONH2 failed to inhibit assembly (Figures 2 and 3A). Secondly,foldamers with more than three lysine or citrulline side chainswere ineffective, encompassing: (Lys-Sal)4-CONH2, (Cit-Sal)4-CONH2, (Lys-Sal)4-COMe, (Lys-Sal)4-COOH and (Lys-Sal)4-COβAla (Figures 2 and 3A). By contrast, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2, which possess threelysine side chains and four aromatic backbone units, were potentinhibitors (Figures 2 and 3A). Thirdly, foldamers with threeor more consecutive Arg side chains were effective inhibitors.Thus, (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2

were potent inhibitors, whereas (Arg-Sal)2-(Cit-Sal)-(Arg-Sal)-CONH2, (Cit-Sal)2-(Arg-Sal)-(Cit-Sal)-CONH2, (Cit-Sal)-(Arg-Sal)-(Cit-Sal)2-CONH2 and (Arg-Sal-Cit-Sal)2-CONH2 wereineffective (Figures 2 and 3A).

Select small molecules that inhibit Aβ42 fibrillization alsodisassemble Aβ42 fibrils [4,57,60]. However, even whenpresent in 4-fold molar excess, (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 did not disassemble Aβ42 fibrils after 24 h (results notshown). Thus, these foldamers do not reverse Aβ42 fibrillization.

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Figure 6 (Arg-Sal)3-(Cit-Sal)-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 inhibit seeded Aβ42 fibrillization

(A–D) Aβ42 (5 μM) was incubated with agitation for 0–2 h at 25◦C without (open squares) or with Aβ42 fibril seed (0.1 μM monomer) in the absence (open circles) or presence of 5 μM (filledtriangles), 10 μM (filled squares) or 20 μM (filled circles) (Arg-Benz)4-CONH2 (A), (Arg-Sal)3-(Cit-Sal)-CONH2 (B), Sal-(Lys-Sal)3-CONH2 (C) or Ac-Sal-(Lys-Sal)3-CONH2 (D). Aβ42 fibrillizationwas assessed by ThT fluorescence. Values represent means +− S.E.M. (n = 3). (E) Aβ42 (5 μM) plus Aβ42 fibril seed (0.1 μM monomer) was incubated with agitation for 4 h at 25◦C plus orminus the indicated foldamer (10 μM). Aβ42 fibrillization was assessed by EM. Scale bar, 500 nm.

Foldamers that inhibit Aβ42 fibrillization do not inhibit NMfibrillization

Next, we assessed foldamer specificity by testing whether they in-hibited amyloidogenesis of the prion domain, NM, of yeast Sup35[63]. (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 did not inhibitNM fibrillization (Figure 5A). In the presence of (Arg-Benz)4-CONH2, NM formed fibrils that exhibited greater ThT fluores-cence (Figure 5A). EM revealed that purely NM fibrils formedin the presence or absence of (Arg-Benz)4-CONH2 and sedi-mentation analysis revealed that equal quantities of NM formedfibrils (results not shown). Thus, (Arg-Benz)4-CONH2 does not

stimulate NM fibrillization. Rather, we suggest that NM accessesa different prion strain in the presence of (Arg-Benz)4-CONH2.NM accesses different prion strains in the presence of certainsmall molecules, such as EGCG [57,63]. None of these foldamersinhibited seeded NM fibrillization (results not shown). Thus, thesefoldamers are not generic inhibitors of amyloidogenesis.

Sal-(Lys-Sal)3-CONH2 delays FUS aggregation

To further test specificity, we assessed inhibition ofaggregation of FUS, an RNA-binding protein with aprion-like domain, which is connected with amyotrophiclateral sclerosis and frontotemporal dementia [1,58,64].

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Figure 7 (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibit spontaneous Aβ43 fibrillization

(A–D) Aβ43 (5 μM) was incubated with agitation for 0–8 h at 25◦C in the absence (open circles) or presence of 5 μM (filled triangles), 10 μM (filled squares) or 20 μM (filled circles)(Arg-Benz)4-CONH2 (A), (Arg-Sal)3-(Cit-Sal)-CONH2 (B), Sal-(Lys-Sal)3-CONH2 (C) or Ac-Sal-(Lys-Sal)3-CONH2 (D). Aβ43 fibrillization was assessed by ThT fluorescence. Values representmeans +− S.E.M. (n = 3). (E) Aβ43 (5 μM) was incubated with agitation for 4 h at 25◦C in the absence or presence of the indicated foldamer (10 μM). Aβ43 fibrillization was assessed by EM.Scale bar, 500 nm.

(Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 did not inhibit FUS aggregation(Figure 5B). Interestingly, Sal-(Lys-Sal)3-CONH2 delayedFUS aggregation (Figure 5B). Sal-(Lys-Sal)3-CONH2 couldserve as a lead foldamer to be optimized against FUS misfolding.

(Arg-Sal)3-(Cit-Sal)-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 inhibitseeded Aβ42 fibrillization

(Arg-Benz)4-CONH2 and Sal-(Lys-Sal)3-CONH2 only inhibitedseeded Aβ42 fibrillization when present at a 4-fold molar excess

over Aβ42 (Figures 6A and 6C, filled circles; Figure 6E).Even at this high concentration, some fibrillization occurredin the presence of (Arg-Benz)4-CONH2 (Figure 6A, filledcircles) but was very limited by Sal-(Lys-Sal)3-CONH2

(Figure 6C, filled circles). Thus, (Arg-Benz)4-CONH2 andSal-(Lys-Sal)3-CONH2 are more potent inhibitors of spontaneousAβ42 fibrillization (Figures 4A and 4C) than seeded Aβ42fibrillization (Figures 6A and 6C). (Arg-Benz)4-CONH2

and Sal-(Lys-Sal)3-CONH2 likely preferentially inhibit therearrangement of Aβ42 oligomers into fibril-nucleatingspecies [22]. Once Aβ42 fibrils have formed,

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Figure 8 Foldamers (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibit seeded Aβ43 fibrillization

(A–D) Aβ43 (5 μM) was incubated with agitation for 0–2 h at 25◦C without (open squares) or with Aβ43 fibril seed (0.1 μM monomer) in the absence (open circles) or presence of 5 μM (filledtriangles), 10 μM (filled squares) or 20 μM (filled circles) (Arg-Benz)4-CONH2 (A), (Arg-Sal)3-(Cit-Sal)-CONH2 (B), Sal-(Lys-Sal)3-CONH2 (C) or Ac-Sal-(Lys-Sal)3-CONH2 (D). Aβ43 fibrillizationwas assessed by ThT fluorescence. Values represent means +− S.E.M. (n = 3). (E) Aβ43 (5 μM) plus Aβ43 fibril seed (0.1 μM monomer) was incubated with agitation for 4 h at 25◦C plus orminus the indicated foldamer (10 μM). Aβ42 fibrillization was assessed by EM. Scale bar, 500 nm.

(Arg-Benz)4-CONH2 and Sal-(Lys-Sal)3-CONH2 have reducedability to inhibit assembly.

Ac-Sal-(Lys-Sal)3-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2

inhibited seeded Aβ42 fibrillization at all concentrations tested(Figures 6B, 6D and 6E). (Arg-Sal)3-(Cit-Sal)-CONH2 wasmore potent with an IC50 of ∼2.5 μM (Figures 6B, 6D and6E). Thus, Ac-Sal-(Lys-Sal)3-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibit Aβ42 fibrillization even after formation of speciesthat nucleate fibrillization.

(Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibitspontaneous Aβ43 fibrillization

It is unknown whether inhibitors that target Aβ42 will alsobe active against Aβ43. In the absence of foldamer, Aβ43fibrillization assembled more rapidly than Aβ42 (Figures 4A–4D, and 7A–7D). Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 did not block spontaneous Aβ43 fibrillization(Figures 7C–7E). Indeed, Sal-(Lys-Sal)3-CONH2 enabled Aβ43

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94 K. M. Seither and others

fibrils to form that exhibited higher ThT fluorescence (Figures 7Cand 7E) and sedimentation analysis revealed that equal quantitiesof Aβ43 formed fibrils (results not shown). Thus, Sal-(Lys-Sal)3-CONH2 does not stimulate Aβ43 fibrillization. Rather, Aβ43may access a different amyloid strain in the presence of Sal-(Lys-Sal)3-CONH2. These findings suggest that potent inhibitorsof spontaneous Aβ42 fibrillization may not inhibit spontaneousAβ43 fibrillization. By contrast, (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 blocked spontaneous Aβ43 fibrillization(Figures 7A, 7B and E). In both cases, small oligomers werethe major species (Figure 7E). The IC50 of (Arg-Sal)3-(Cit-Sal)-CONH2 was ∼3.1 μM (Figures 7A and 7B).

(Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibit seededAβ43 fibrillization

Aβ43 fibrils eliminated the lag phase of Aβ43 assembly(Figures 8A–8D, compare open squares and open circles). Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 did not inhibitseeded Aβ43 fibrillization (Figures 8C–8E). Sal-(Lys-Sal)3-CONH2 enabled Aβ43 to access fibrillar forms that generateda higher ThT fluorescence signal, perhaps indicative of a distinctAβ43 amyloid strain (Figure 8C). By contrast, (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 blocked seeded Aβ43fibrillization (Figures 8A, 8B and 8E). The IC50 of (Arg-Sal)3-(Cit-Sal)-CONH2 against seeded Aβ43 fibrillization was ∼1.7 μM.

Foldamers inhibit Aβ42 and Aβ43 fibrillization under differentassembly conditions

Next, we established that foldamers inhibited spontaneous andseeded Aβ42 and Aβ43 fibrillization under different assemblyconditions, which might support formation of different amyloidstrains. Thus, we avoided DMSO in Aβ preparation andassembled in a higher pH buffer. Under these conditions, anegative control foldamer, (Cit-Sal)4-CONH2, had no effect(Figure 9). By contrast, (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 inhibited spontaneous and seeded Aβ42 fibrillization(Figure 9), whereas only (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 inhibited spontaneous and seeded Aβ43fibrillization (Figure 9).

(Arg-Sal)3-(Cit-Sal)-CONH2 antagonizes formation of A11-reactiveAβ42 and Aβ43 oligomers

Could foldamers inhibit the formation of toxic Aβ42 and Aβ43oligomers? To assess toxic Aβ42 and Aβ43 oligomer formation,we employed the conformation-specific A11 antibody, whichspecifically recognizes preamyloid oligomers formed by multipleproteins, including Aβ42, but not monomers or fibrils [21].We assessed formation of A11-reactive species at the start ofspontaneous assembly (0 h), at the end of lag phase (0.5 h), andat the endpoint of fibrillization (4 h). In the absence of Aβ42and Aβ43, no A11 immunoreactivity was observed (results notshown). For Aβ42 and Aβ43, A11-reactive conformers werescarce at the start of the reaction (Figure 10A, buffer controls,black bars), abundant at end of lag phase (Figure 10A, buffercontrols, grey bars), and declined once fibrillization was complete(Figure 10A, buffer controls, white bars). Aβ43 exhibited greaterA11-immunoreactivity than Aβ42 and appears more prone toaccessing this toxic conformation (Figure 10A).

A negative control foldamer, (Cit-Sal)4-CONH2 (Figure 2), hadno effect on the appearance and disappearance of A11-reactive

Figure 9 Foldamers inhibit Aβ42 and Aβ43 fibrillization under differentassembly conditions

Aβ42 or Aβ43 (5 μM) were incubated with agitation for 16 h at 25◦C without orwith Aβ42 fibril seed or Aβ43 fibril seed (0.1 μM monomer) plus or minus 20 μM(Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2, Ac-Sal-(Lys-Sal)3

-CONH2 or (Cit-Sal)4-CONH2. Fibrillization was assessed by ThT fluorescence. Values representmeans +− S.E.M. (n = 3).

Aβ42 and Aβ43 conformers (Figure 10A). (Arg-Benz)4-CONH2,Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 had noeffect on the abundance of A11-reactive Aβ42 or Aβ43 oligomersafter 0.5 h (Figure 10A, grey bars). Thus, these foldamers inhibitspontaneous Aβ42 or Aβ43 fibrillization without affecting theformation of A11-reactive conformers. Furthermore, after 4 h inthe presence of (Arg-Benz)4-CONH2, Sal-(Lys-Sal)3-CONH2 andAc-Sal-(Lys-Sal)3-CONH2, A11-reactive Aβ42 species remainedat higher levels and did not decline as much as they did inthe absence of foldamer (Figure 10A, white bars). Thus, (Arg-Benz)4-CONH2, Sal-(Lys-Sal)3-CONH2, and Ac-Sal-(Lys-Sal)3-CONH2 stabilize A11-reactive conformers. (Arg-Benz)4-CONH2,but not Sal-(Lys-Sal)3-CONH2 or Ac-Sal-(Lys-Sal)3-CONH2, hada similar effect on A11-reactive Aβ43 species (Figure 10A). Bycontrast, A11-reactive Aβ43 species declined more extensivelyafter 4 h in the presence of Sal-(Lys-Sal)3-CONH2 or Ac-Sal-(Lys-Sal)3-CONH2 (Figure 10A, white bars), which do not inhibitspontaneous Aβ43 fibrillization (Figures 7C and 7D).

(Arg-Sal)3-(Cit-Sal)-CONH2 inhibited the formation of A11-reactive Aβ42 and Aβ43 conformers after 0.5 h (Figure 10A,grey bars). After 4 h, (Arg-Sal)3-(Cit-Sal)-CONH2 preventedfurther accumulation of A11-reactive Aβ42 and Aβ43 conformers(Figure 10A, white bars). Thus, (Arg-Sal)3-(Cit-Sal)-CONH2

inhibits fibrillization as well as toxic oligomer formation by Aβ42and Aβ43. (Arg-Sal)3-(Cit-Sal)-CONH2 might inhibit Aβ42 andAβ43 misfolding by a mechanism that is distinct to the otherfoldamers and arrests Aβ42 and Aβ43 misfolding prior to anA11-reactive oligomeric state.

(Arg-Sal)3-(Cit-Sal)-CONH2 inhibits formation of toxic Aβ42 andAβ43 conformers

Next, we evaluated the relative toxicity of Aβ42 and Aβ43conformers formed in the absence or presence of foldamers. Weapplied Aβ42 and Aβ43 conformers to SH-SY5Y neuroblastomacells and assessed cell viability using MTT reduction and LDHrelease. Foldamers and buffer display little toxicity in the absence

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Specific aromatic foldamers antagonize Aβ amyloidogenesis 95

Figure 10 (Arg-Sal)3-(Cit-Sal)-CONH2 inhibits formation of toxic Aβ42 and Aβ43 conformers

(A–C) Aβ42 or Aβ43 (5 μM) was incubated at 25◦C with agitation for 0 h (black bars), 0.5 h (grey bars) or 4 h (white bars) in the absence or presence of 20 μM (Arg-Benz)4-CONH2,(Arg-Sal)3-(Cit-Sal)-CONH2, Sal-(Lys-Sal)3-CONH2, Ac-Sal-(Lys-Sal)3-CONH2 or (Cit-Sal)4-CONH2. At the indicated times, the amount of A11-reactive species present (A) or toxicity toSH-SY5Y neuroblastoma cells in culture was determined via MTT reduction (B) or LDH release (C). We also assessed the toxicity of buffer, (Arg-Benz)4-CONH2, (Arg-Sal)3-(Cit-Sal)-CONH2,Sal-(Lys-Sal)3-CONH2, Ac-Sal-(Lys-Sal)3-CONH2 or (Cit-Sal)4-CONH2 alone (B and C). Values represent means +− S.E.M. (n = 3). A one-way ANOVA with the post-hoc Dunnett’s multiplecomparisons test was used to compare Aβ42 plus buffer to each Aβ42 plus foldamer condition (* denotes P < 0.05). Likewise, a one-way ANOVA with the post-hoc Dunnett’s multiple comparisonstest was used to compare Aβ43 plus buffer to each Aβ43 plus foldamer condition (* denotes P < 0.05).

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of Aβ (Figures 10B and 10C, far right). In the absence of foldamer,Aβ42 and Aβ43 exhibited little toxicity after 0 h (Figures 10Band 10C), consistent with reduced A11 immunoreactivity at thistime (Figure 10A). Aβ42 and Aβ43 were more toxic after 0.5 hof assembly than after 4 h (Figures 10B and 10C), indicatingthat conformers that accumulate at the end of lag phase aremore toxic than mature fibrils. In the absence of foldamer, Aβ43conformers were generally more toxic than Aβ42 conformers(Figures 10B and 10C). The negative control foldamer, (Cit-Sal)4-CONH2, had no effect on toxicity (Figures 10B and 10C).(Arg-Benz)4-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2 had no effect on the toxicity of Aβ42 conformersafter 0.5 h of assembly (Figures 10B and 10C, grey bars), butafter 4 h of assembly the toxicity of Aβ42 conformers wasenhanced (Figures 10B and 10C, white bars). Thus, (Arg-Benz)4-CONH2, Sal-(Lys-Sal)3-CONH2 and Ac-Sal-(Lys-Sal)3-CONH2

inhibit spontaneous Aβ42 fibrillization such that more toxicconformers are maintained (Figures 10A–C). For Aβ43, neitherSal-(Lys-Sal)3-CONH2 nor Ac-Sal-(Lys-Sal)3-CONH2 affectedthe toxicity of conformers after 0.5 h or 4 h (Figures 10B and 10C).However, as for Aβ42, (Arg-Benz)4-CONH2 had no effect on thetoxicity of Aβ43 conformers after 0.5 h of assembly (Figures 10Band 10C, grey bars), but after 4 h the toxicity of Aβ43 conformerswas enhanced (Figures 10B and 10C, white bars). Thus, (Arg-Benz)4-CONH2 inhibits spontaneous Aβ43 fibrillization in amanner that maintains toxic conformers (Figures 10A–10C).

(Arg-Sal)3-(Cit-Sal)-CONH2, which inhibited the formation ofA11-reactive Aβ42 and Aβ43 conformers after 0.5 h (Figure 10A,grey bars), also partially reduced the toxicity of Aβ42 and Aβ43conformers at this time (Figures 10B and 10C, grey bars) and at4 h (Figures 10B and 10C, white bars). Although Aβ42 and Aβ43conformers still conferred toxicity in comparison with buffercontrols, (Arg-Sal)3-(Cit-Sal)-CONH2 was the only foldamer thatantagonized Aβ42 and Aβ43 toxicity.

(Arg-Sal)3-(Cit-Sal)-CONH2 inhibits spontaneous and seededAβ42 and Aβ43 fibrillization and reduces accumulation of toxicAβ42 and Aβ43 conformers. This combination of propertiescould have therapeutic potential for three reasons. First, (Arg-Sal)3-(Cit-Sal)-CONH2 antagonizes Aβ42 as well as Aβ43, whichis an often overlooked but highly toxic Aβ species [13–16].Secondly, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibits the formationof toxic Aβ42 and Aβ43 conformers, which could reducelocalized neurodegeneration [65]. Thirdly, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibits seeded Aβ42 and Aβ43 assembly, which couldprevent the spreading of Aβ pathology throughout the brain inAD [29–31]. Further studies are needed to assess the utility of(Arg-Sal)3-(Cit-Sal)-CONH2 against Aβ misfolding and toxicityin the metazoan nervous system.

Future studies will reveal the mechanisms by which foldamersantagonize Aβ-misfolding. Foldamers have amides orientedappropriately (Figure 2) to block growth from fibril endsduring seeded polymerization. They are also relatively flat andaromatic (Figure 2) and might antagonize secondary nucleationby binding to the lateral surface of fibrils. Foldamer insertion intomolten oligomers could inhibit rearrangement events required fornucleation during spontaneous assembly. Differences in the abilityof specific foldamers to inhibit Aβ42 fibrillization comparedwith Aβ43 fibrillization probably reflect differential antagonismof events driven by the additional C-terminal steric zipperhexapeptide (G38VVIAT43) of Aβ43.

Aromatic foldamers could be useful amyloidogenesis inhibitorsfor various disease-associated proteins. Indeed, another classof aromatic foldamer inhibits amylin fibrillization, which isconnected to Type 2 diabetes [66]. Thus, foldamers await furtherdevelopment to antagonize protein misfolding in several settings.

AUTHOR CONTRIBUTION

Conceived and designed the experiments: Katelyn Seither, Heather McMahon, NikitaSingh, Hejia Wang, Mimi Cushman-Nick, Geronda Montalvo, William DeGrado and JamesShorter. Performed the experiments: Katelyn Seither, Heather McMahon, Nikita Singh, HejiaWang, Mimi Cushman-Nick and James Shorter. Analysed the data: Katelyn Seither, HeatherMcMahon, Nikita Singh, Hejia Wang, Mimi Cushman-Nick, Geronda Montalvo, WilliamDeGrado and James Shorter. Contributed key reagents/materials: Geronda Montalvo andWilliam DeGrado. Wrote the paper: Katelyn Seither, William DeGrado and James Shorter.

FUNDING

This work was supported by the National Institutes of Health [grant numbers T32AG000255and F31NS067890 (to M.C.N.), GM54616 (to W.F.D.), DP2OD002177 (to J.S.),R21NS067354 (to J.S.), R21HD074510 (to J.S.) and R01GM099836 (to J.S.)]; NationalScience Foundation Materials Research Science and Engineering Centers grant to theLaboratory for Research on the Structure of Matter of the University of Pennsylvania[grant number DMR-1120901 (to W.F.D.)]; Muscular Dystrophy Association ResearchAward [grant number MDA277268]; Packard Center for ALS Research at Johns HopkinsUniversity, Target ALS and an Ellison Medical Foundation New Scholar in Aging Award(to J.S.)].

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Received 6 December 2013/20 August 2014; accepted 21 August 2014Published as BJ Immediate Publication 21 August 2014, doi:10.1042/BJ20131609

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